1. Field of the Invention
This invention relates to a process for the determination of carbon dioxide in a sample, such as a sample of blood serum, and more specifically to a pair of coupled enzymatic reactions for such a determination.
2. Discussion of the Prior Art
The human body generates a large amount of carbon dioxide; only a small portion of which is reutilized, for example, in urea formation or other carboxylation reactions. The rest must be eliminated. One way in which carbon dioxide is eliminated is through the blood stream, and the concentration of carbon dioxide in the blood stream has a profound effect on body function. A moderate elevation in the concentration of carbon dioxide in the blood supply to the brain, for example, greatly enhances cerebral circulation. Abnormal concentrations of carbon dioxide in the blood stream, then, are either the product of or in some circumstances, the cause of a variety of illnesses. For this reason, the measurement of carbon dioxide content in the blood stream or other body fluids is an important measurement in medical diagnostics.
Only a small portion of the carbon dioxide (CO.sub.2) introduced into the blood stream remains in the physically dissolved state. The rest is catalyzed into carbonic acid (H.sub.2 CO.sub.3) by carbonic anhydrase, according to the reaction ##STR1## and the carbonic acid is converted into hydrogen ions and bicarbonate ion (HCO.sub.3.sup.-) according to the reaction EQU H.sub.2 CO.sub.3 .revreaction. H.sup.+ + HCO.sub.3.sup.- .
The term "carbon dioxide content" as it applies to body fluid, actually means the sum of bicarbonate ions, carbonic acid and dissolved carbon dioxide.
The carbonic anhydrase enzyme reaction discussed above has been used to measure the carbon dioxide content of carbonated beverages by adding the enzyme to catalyze the production of carbonic acid and measuring the amount of carbonic acid produced by some indicator, but due to the presence of carbonic acid and the enzyme carbonic anhydrase itself in body fluids, such a process is not appropriate for the measurement of the carbon dioxide content of body fluids. The measurement of the carbon dioxide content in such fluids is usually performed by three basic approaches. In the gasometric approach, CO.sub.2 gas is liberated from the serum by the addition of acid, and the volume of CO.sub.2 evolved is measured manometrically or volumetrically. Since bicarbonate is a base and produces a pH change when added to weakly buffered solutions, a second approach is to measure the amount of bicarbonate in a sample by measuring the color change in a buffered solution by using an indicator such as phenolphthalein or phenol red. Finally, the partial pressure of CO.sub.2 in blood can be measured with a CO.sub.2 electrode and the carbon dioxide content can be calculated from this partial pressure if the pH is also measured.
Except for the indicator method, these processes are difficult to automate, especially for use in instruments designed to perform a number of tests by measuring the change in optical absorbance of the test sample. The indicator method, discussed above, suffers from the fact that it must be calibrated every time the measurement is made due to changes in the indicator solution with time. There is need, therefore, for a rapid, reliable, precise process for measuring the carbon dioxide content in a sample, particularly a sample of body fluid, by a method that can be easily automated.
The present invention provides such a process. In its broadest aspect, it comprises the use of one of three coupled enzyme reactions. The preferred reaction is: ##STR2## where PEP carboxylase stands for phosphoenol pyruvate carboxylase, P.sub.i stands for inorganic phosphorus, MDH stands for malate dehydrogenase, and NADH and NAD, respectively, stand for the reduced and oxidized form of nicotinamide adenine dinucleotide.
In some circumstances, however, the first of the two reactions given above can be replaced by ##STR3## or a reaction involving phosphoenol pyruvate carboxy kinase and a nucleotide diphosphate such as ##STR4## where ADP stands for adenosine disphosphate and ATP stands for adenosine triphosphate, or the following reaction involving phosphoenol pyruvate carboxyltransphorylase and inorganic phosphorous: ##STR5## where PP.sub.i stands for inorganic pyrophosphate.
The process comprises mixing measured amounts of body fluid with excess amounts of a substrate/enzyme combination selected from the group consisting of
a. pyruvate and a nucleotide triphosphate/pyruvate carboxylase, PA1 b. phosphoenol pyruvate/phosphoenol pyruvate carboxylase, PA1 c. phosphoenol pyruvate and a nucleotide disphosphate/phosphoenol pyruvate carboxy kinase, and PA1 d. phosphoenol pyruvate and inorganic phosphorous/puruvate carboxyltransphorylase, PA1 0.01 M phosphate PA1 5.00 mM aspartate PA1 (pH 7.8) PA1 0.01 M phosphate PA1 5.0 mM aspartate PA1 1.0 mM DTE PA1 (pH 7.8) PA1 10% ethanol, PA1 2.5 mM aspartate PA1 0.1 M phosphate PA1 2.5 mM aspartate PA1 25% glycerol PA1 1.0% albumin PA1 (pH 7.8) PA1 50% glycerol PA1 2.5 mM aspartate PA1 .01 m phosphate PA1 1.0 mM MgCl.sub.2 PA1 1.0 mM DTE PA1 (pH 7.8) PA1 2.05 M (NH.sub.4).sub.2 SO.sub.4 PA1 2.5 mM aspartate
and excess amounts of malate dehydrogenase and the reduced form of nicotinamide adenine dinucleotide; and determining the change in the concentration of the reduced form of nicotinamide adenine dinucleotide in the mixture so formed while maintaining the system at a substantially constant temperature and a substantially constant pH.
The term body fluid, as used in this context, means any liquid containing natural products of the body such as an actual body fluid (blood) or a reconstituted body substance (serum).
Each of the above reactions, which actually measure the bicarbonate concentration in a fluid, can be used as a measure of the carbon dioxide content in the fluid because of the equilibrium which exists between CO.sub.2 in solution, bicarbonate and carbonic acid. It is well known, however, that carbonic anhydrase will act to convert dissolved CO.sub.2 into bicarbonate. See for example the article entitled The Carboxylation of Phosphoenolpyruvate and Pyruvate by T. G. Cooper et al. in The Journal of Biological Chemistry, 243, 3857, 1968. For this reason, the accuracy of the test procedures discussed above can be increased by the addition of carbonic anhydrase to the test solution.
Furthermore, each of the reactions discussed above proceed more efficiently if a metal ion cofactor for the enzyme used is present in the reaction mixture to act as a catalyst. Magnesium ions (Mg.sup.+.sup.+) or manganese ions (Mn.sup.+.sup.+) appear to be common metal ion cofactors in all cases discussed above.
Since the reduced form of nicotinamide adenine dinucleotide absorbs light very strongly between about 290 and about 380 millimicrons, preferably at 340 millimicrons (nm.) while the oxidized form does not, the rate of disappearance of the reduced form is directly proportional to the decrease in absorbance of light at this wavelength and at constant temperature, usually at a constant temperature between 15.degree. and 50.degree.C., and a constant pH, usually between about 7.5 and about 10.5; and can be measured readily by those skilled in the art using a conventional spectrophotometric procedure. Since the rate of oxidation of the reduced form of nicotinamide adenine dinucleotide is also proportional to the rate of formation of oxaloacetate and the rate of formation of oxaloacetate is a function of the concentration of bicarbonate in the system, the decrease in absorbance at 340 millimicrons can be used as a direct measure of the original concentration of bicarbonate in the sample fluid. It should also be understood that other wavelengths, e.g., 366 millimicrons, can be used for the foregoing purpose. Furthermore, the enzyme systems can be coupled with a redox-dye system which will change the wavelength at which the absorbance measurement can be made.
Since blood serum often contains lactate dehydrogenase (LDH) for which pyruvate is a substrate, the process involving pyruvate and pyruvate carboxylase can give an erroneous reading because of the interference provided by this competing reaction, unless the LDH is removed from the sample, or the competing reaction inhibited in some way. For this reason, the reaction involving phosphoenol pyruvate and PEP carboxylase is preferred over that involving pyruvate and pyruvate carboxylase. It is also preferred over the reaction involving phosphenol pyruvate carboxy kinase and a nucleotide diphosphate because it is a simpler reaction involving fewer reagents.
Except for the difference in the compound and its enzyme, the reactions are quite similar. For convenience, the discussion which follows will be limited to the preferred embodiment involving phosphoenol pyruvate and phosphoenol pyruvate carboxylase. As far as this reaction is concerned, it is known that phosphoenol pyruvate carboxylase catalyzes the carboxylation of phosphoenol pyruvate by bicarbonate to oxaloacetate and inorganic phosphate. This reaction has, in fact, been used in conjunction with the malic dehydrogenase indicator reaction to assay the activity of PEP carboxylase, by adding an excess amount of bicarbonate. See Escherichia coli Phosphoenolpyruvate Carboxylase Characterization and Sedimentation Behavior by T. E. Smith in the Archives of Biochemistry and Biophysics, 128, 611, (1968). Use has also been made of these two coupled reactions to measure the carbon dioxide concentration in reagents. This latter determination was an end point determination, however, which was allowed to go to completion under a layer of mineral oil.
These coupled reactions, however, have not been used to measure the carbon dioxide content of body fluids. In particular, they have never been used to measure carbon dioxide content by a rate determination. Furthermore, because of the presumed lack of stability of the enzymes used in the determination, and the sensitivity of the process to carbon dioxide which is absorbed into the body fluid from the air rather than from body functions, such processes have generally been discounted for use in automated analysis. I have discovered that an accurate measure of the carbon dioxide content of body fluid can be made using the coupled reactions discussed above. In particular, I have discovered that a rapid and accurate measurement of the carbon dioxide content can be made by using excess non-rate limiting amounts of all constituents, except the body fluid and phosphoenopyruvate carboxylase, and measuring the rate at which the absorbancy of the solution so formed decreases due to oxidation of NADH. I have also discovered that the measurements can be made without fear of inaccuracy introduced by the ambient carbon dioxide level of the air and water if most of the reagents are added to the body fluid in solid form. These solids reagents can be made using a freeze-dry or dry blending technique. Finally, I have discovered that phosphoenol pyruvate carboxylase can be stabilized by the various techniques discussed below.
The production of PEP carboxylase can be carried out according to the techniques described in the article by J. L. Canovas et al. in Biochem Biophys Acta, 96, 189 (166) entitled Properties and Regulation of Phosphopyruvate Carboxylase Activity in Escherietia coli, and purified as disclosed in the article by T. E. Smith (supra). In particular, 209 grams of E. Coli cells, which had been grown in a Kornberg glucose salt medium, were suspended (30% w/v) in a buffer solution, at a pH of 8.0, composed of 5.0 mM Tris, 1.0 mM magnesium chloride, and 1.0 mM dithioerythritol (DTE). The cells were disrupted by 5 minutes of sonication and the cell debris was removed by centrifugation.
The supernatant, about 500 ml., was pooled and 34 ml. of a 2% solution of protamine sulfate in 5 mM acetate buffer at a pH of 5.0 was added. The precipitate was removed by centrifugation and the supernatant was made 0.4 saturated with (NH.sub.4).sub.2 SO.sub.4 by addition of 116 g. of (NH.sub.4).sub.2 SO.sub.4. The precipitate was removed by further centrifugation and the supernatant was brought to 0.50 saturation with (NH.sub.4).sub.2 SO.sub.4. This precipitate was collected by centrifugation and then dissolved in 15 mls. Tris-Mg-DTE buffer and dialyzed for 12 hours against 2 liters of buffer.
The dialyzed solution was then pumped onto a DEAE cellulose column (2.5 .times. 40 cm column bed) using a peristaltic pump to maintain a constant flow rate. When the sample was on the column, the column was eluted with a linear chloride gradient by running 0.75 N potassium chloride into the Tris-Mg-DTE buffer. Seven milliliter fractions were collected. The enzyme came off the DEAE cellulose column at a chloride concentration of 0.16 to 0.19 M, and the fractions between 35 and 50 contained a substantial amount of highly active PEP carboxylase.
The enzyme phosphoenol pyruvate carboxylase is an allosteric enzyme and is activated by a number of compounds. Acetylcoenzyme A is the most effective activator however several others, including fructose 1,6 diphosphate, and organic solvents such as ethanol, methanol, propanol and 1,4 dioxane are effective. The reaction mixture should also contain effective amounts of one of those activators.
One of the requirements for the kinetic determination of a substrate is that the enzyme is stable, i.e., the enzyme shows little or no change in activity within the time span of the determination. Furthermore, it is desirable that the enzyme be stable for a reasonable time so that the test material will have a reasonable shelf-life. Studies have shown that the following compositions are useful as stabilizing agents for the enzyme PEP carboxylase.
a.
b.
c.
d.
e.
f.
Table I shows the effect of these stabilizing agents on compositions studied at 4.degree. and 25.degree.C.
TABLE I __________________________________________________________________________ DAYS STABILIZING TEMP AGENT .degree.C 0 1 2 4 7 10 12 14 17 22 __________________________________________________________________________ % ACTIVITY REMAINING (a) 4.degree. 100 98 98 96 97 97 93 89 25.degree. 100 79 4 2 (b) 4.degree. 100 98 101 102 101 97 92 91 25.degree. 100 85 43 17 (c) 4.degree. 100 100 103 105 98 100 100 95 96 25.degree. (d) 4.degree. 100 100 103 101 101 98 100 100 94 25.degree. 100 85 25 8 (e) 4.degree. 100 100 99 98 97 99 96 96 96 25.degree. 100 98 79 64 47 (f) 4.degree. 100 102 100 105 101 100 103 104 109 25.degree. 100 98 99 97 91 __________________________________________________________________________
All of the compounds have a stabilizing effect, particularly at low temperature storage. The last two, (e) and (f), however, have a marked effect even at room temperature. Preferably, the mixture containing PEP carboxylase will also contain effective amounts of one of these stabilizing agents.
Since carbon dioxide in the atmosphere will permeate the reagents used in the present process and will be converted to bicarbonate as discussed above, for an accurate determination of the carbon dioxide content of body fluid, some attempt must be made to insure that the reagents used are free from atmospheric carbon dioxide. I have found that a blanket of CO.sub.2 free nitrogen is effective in reducing CO.sub.2 absorption by the solution used in the reaction. Where this is impractical, the buffer and reagents are added as solids, and in order to avoid the problems of CO.sub.2 contamination of the reaction solution, the reagents were added as solids to the reaction solution immediately before the reaction.
The reagents used in the assays described in Examples II and III below were prepared as follows:
Malate Dehydrogenase Solution
One ml. of malate dehydrogenase solution (10 mg/ml) was added to 199 ml. of 50% glycerol solution. Fifty microliters of this solution is used per assay.
Tris-Tris-HCl/Mg Buffer Tablets
Tris-Hydroxyaminomethane and Tris Hydroxyaminomethane hydrochloride and MgSO.sub.4 . 7H.sub.2 O were sized to -30, to +60 U.S. Standard mesh size. The Tris, Iris HCl and MgSO.sub.4 were blended together in a stainless steel pan with a spatula. Polyethylene glycol (PEG 2000) was blended in by hand. This mixture was passed through a No. 20 U.S. Standard mesh screen to disperse the PEG, and then through a No. 30 mesh screen two times to reduce particle size and increase fines content. Eighty milligram tablets were prepared on a Stokes rotary tablet press. Each tablet contained on the average of 43 mg Tris, 22.5 mg Tris HCl, 10.8 mg MgSO.sub.4 and 4.1 mg PEG. One tablet in 5 ml water at 37.degree.C. produces a pH of 8.5.
NADH Tablets
In order to achieve a precise amount of NADH in a tablet, it was necessary to freeze spray the NADH using a Freon Fluorocarbon refrigerant. This requires putting the NADH in an alkaline mannitol solution and spraying. Because of the problem of CO.sub.2 pickup by alkaline solutions, the solution was prepared and sprayed under nitrogen pressure. Mannitol (58.40 g) and NADH (disodium salt) (1.60g) were dissolved in 240 ml of purified water under a blanket of nitrogen. When dissolved, the vessel was pressurized with nitrogen and the solution was sprayed into a bath of Freon. The frozen material was collected and lyophilized for 48 hours in a Virtis freeze dryer. The product (47.1 g) was blended with 2.4 g of PEG and 47-48 mg tablets were prepared containing 1.1 mg NADH. One tablet per assay was used.
Phosphoenol Pyruvate Tablets
Phosphoenol pyruvate monocyclohexylammonium salt was blended with Tris, mannitol, PEG in the following amounts: 18 g PEP; 27.6 g Tris; 68.4 g mannitol and 6 g PEG to produce a 100 mg tablet containing 15 mg PEP monocyclohexylammonium salt. One tablet per assay was used.
Phosphoenol Pyruvate Carboxylase Solution
Phosphoenol pyruvate carboxylase was isolated as described above. In Example II, the enzyme was not purified past the ammonium sulfate 30-50% fractionation stage. To 56 ml. of the 30-50% ammonium sulfate cut (26 IU PEP carboxylase/ml.), in the phosphate-asparatate buffer described above, was added 13.5 mg MgSO.sub.4 . 7H.sub.2 O and 17.3 mg dithioerythritol. To this solution was added 56 ml glycerol. Sixty-five microliters of this solution was used per assay. This is approximately 0.85 IU per assay.